Construction and visualization of GFP-tyrosinase in mouse cells

초록

In many regions of the world, having a light and even skin color is highly valued. Skin pigmentation results in part from the transfer of melanized melanosomes by melanocytes to neighboring keratinocytes. Tyrosinase plays the key role in melanin synthesis. PKCβ activates tyrosinase directly by phosphorylating couple of serine residues in the cytoplasmic domain of this protein. To provide a dynamic method of visualizing the tyrosinase in melanosome, we generated fluorescent fusion proteins by using the green fluorescent protein (GFP) and red fluorescent protein (RFP) as fluorescent markers at the carboxyl termini and the amino termini of this enzyme. The intracellular localization of tyrosinase was then examined by fluorescence and confocal microscopy after transient transfection of the expression vector into Melan-A cells. Results suggest that GFP and RFP fusion tyrosinase constructs are useful to visualize tyrosinase in mouse cells. This system might be useful to study molecular mechanisms of PKC-mediated tyrosinase activation in melanocytes.